Curation Information

Publication: Analyses of binding sequences of the two LexA proteins of Xanthomonas axonopodis pathovar citri.;Yang MK, Hsu CH, Sung VL;Molecular genetics and genomics : MGG 2008 Jul; 280(1):49-58 [18437426]
TF: LexA [Q8PN77, view regulon]
Reported TF sp.: Xanthomonas axonopodis pv. citri XW47
Reported site sp.: Xanthomonas axonopodis pv. citri XW47
Created by: Dinara Sagitova
Curation notes: -

Experimental Process

Determination of the LexA2-binding site within the X. axonopodis pv. citri lexA2 promoter region was done by using DNA probes corresponding to different regions of the promoter. The results of this analysis indicated that LexA2 binds to a region located between −47 and −22 relative to the lexA2 translation start site. LexA2 binding to this site was confirmed by performing EMSAs. A palindromic sequence was found in this promoter region. Binding specificity was confirmed with site-directed mutagenesis and EMSA analyses. Luciferase reporter assays performed with either the wild-type or mutated lexA2 promoter indicated that LexA2 represses lexA2 expression in X. axonopodis pv. citri.

Transcription Factor Binding Sites

Sites reported in the paper
Sites matched in the genome

TGTACAAATGTACA

Gene Regulation

Regulated genes for each binding site are displayed below. Gene regulation diagrams show binding sites, positively-regulated genes, negatively-regulated genes, both positively and negatively regulated genes, genes with unspecified type of regulation. For each indvidual site, experimental techniques used to determine the site are also given.

Site sequence	Regulated genes	Gene diagram	Experimental techniques	TF function	TF type
TGTACAAATGTACA	lexA,		Experimental technique details EMSA (ECO:0001807) EMSA - ECO:0001807 Electro-mobility shift-assays (or gel retardation assays) are a standard way of assessing TF-binding. A fragment of DNA of interest is amplified and labeled with a fluorophore. The fragment is left to incubate in a solution containing abundant TF and non-specific DNA (e.g. randomly cleaved DNA from salmon sperm, of all things) and then a gel is run with the incubated sample and a control (sample that has not been in contact with the TF). If the TF has bound the sample, the complex will migrate more slowly than unbound DNA through the gel, and this retarded band can be used as evidence of binding. The unspecific DNA ensures that the binding is specific to the fragment of interest and that any non-specific DNA-binding proteins left-over in the TF purification will bind there, instead of on the fragment of interest. EMSAs are typically carried out in a bunch of fragments, shown as multiple double (control+experiment) lanes in a wide picture. Certain additional controls are run in at least one of the fragments to ascertain specificity. In the most basic of these, specific competitor (the fragment of interest or a known positive control, unlabelled) is added to the reaction. This should sequester the TF and hence make the retardation band disappear, proving that the binding is indeed specific - Experimental technique details Luciferase reporter assay (ECO:0005648) Luciferase reporter assay - ECO:0005648 A type of reporter assay . The idea is identical to a betagalactosidase assay, but here fluorescence of luciferase gene product is used as the reporter. - Experimental technique details Site directed mutagenesis (ECO:0005667) Site directed mutagenesis - ECO:0005667 Target-specific mutation, as opposed to non-specific mutation. In the context of TF-binding sites, site-directed mutagenesis is typically used to establish/confirm the specific sequence and location of a site, often in tandem with EMSA. Different positions of a putative binding site are mutated to non-consensus (or random) bases and binding to the mutated site is evaluated through EMSA or other means. Often implemented only in conserved motif positions or serially through all positions of a site. - Experimental technique details Visual sequence inspection (nan) Visual sequence inspection - nan Visual inspection of promoter region to identify putative binding sites based on previous knowledge -	repressor	not specified